Cloning,Expression, and Characterization of Rice α-Galactosidase

We have successfully cloned an α-galactosidase gene from a rice cDNA library and transformed it into Escherichia coli BL21. It was subsequently cloned to the pPIC9K vector and expressed in Pichia pastoris. A selected clone was found to result in high production yield of the galactosidase enzyme. The secreted enzyme was purified, and it revealed as a major protein band on an SDS-PAGE gel. The optimal pH value, enzyme stabilities, and substrate specificity were studied. The enzyme specificity toward the terminal α1→6, 1→4, and 1→3 linked galactosyl residue from various substrates was investigated. By determining the Michelis constant (Km) of the enzyme for melibiose, raffinose, and stachyose, our results showed that melibiose was hydrolyzed faster than raffinose, whereas the published data reported a reversed sequence, raffinose > melibiose. The enzyme also showed the ability of converting B red blood cells into O red cells. The objective of this work is to develop the Pichia system to produce a large quantity of enzyme for blood cell conversion for transfusion.     

Rice Research： Past, Present and Future

Rice （Oryza sativa L.） is a major crop in the world and provides the staple food for over half of the world＇s population. From thousands of years of cultivation and breeding to recent genomics, rice has been the focus of agriculture and plant research.     

Silicon-Mediated Resistance in a Susceptible Rice Variety to the Rice Leaf Folder,Cnaphalocrocis medinalis Guenée (Lepidoptera: Pyralidae)

The rice leaf folder, Cnaphalocrocis medinalis (Guenée), is one of the most destructive rice pests in Asian countries. Rice varieties resistant to the rice leaf folder are generally characterized by high silicon content. In this study, silicon amendment, at 0.16 and 0.32 g Si/kg soil, enhanced resistance of a susceptible rice variety to the rice leaf folder. Silicon addition to rice plants at both the low and high rates significantly extended larval development and reduced larval survival rate and pupation rate in the rice leaf folder. When applied at the high rate, silicon amendment reduced third-instars’ weight gain and pupal weight. Altogether, intrinsic rate of increase, finite rate of increase and net reproduction rate of the rice leaf folder population were all reduced at both the low and high silicon addition rates. Although the third instars consumed more in silicon-amended treatments, C:N ratio in rice leaves was significantly increased and food conversion efficiencies were reduced due to increased silicon concentration in rice leaves. Our results indicate that reduced food quality and food conversion efficiencies resulted from silicon addition account for the enhanced resistance in the susceptible rice variety to the rice leaf folder.     

Studies on α-Glucosidase in Rice

Two kinds of αglucosidase which were homogeneous in disc electrophoretic and ultra-centrifugal analysis were isolated from rice seeds by means of ammonium sulfate fractionation and CM-cellulose, Sephadex G–100 and DEAE-cellulose column chromatography and designated as α-glucosidase I and α-glucosidase II.

Both α-glucosidases hydrolyzed maltose and soluble starch to glucose and showed same optimal pH (4.0) on the both substrates. In addition, both enzymes acted on various α-linked gluco-oligosaccharides and soluble starch but little or not on α-linked hetero-glucosides and α-l,6-glucan (dextran).

Activity of the enzymes on maltose and soluble starch was inhibited by Tris and erythritol. α-Glucosidase II was more sensitive to the inhibitors than α-glucosidase I.

Km value for maltose was 1.1 mM for α-glucosidase I and 2.0 mM for α-glucosidase II.     

Absorption,Translocation and Metabolism of O,O-Diisopropyl S-Benzyl Phosphorothiolate (Kitazin P®) in Rice Plant

Behavior and metabolism of O,O-diisopropyl S-benzyl phosphorothiolate (Kitazin P©) in rice plant were examined using ³²P, ³⁵S-double labeled compound. Uptake of Kitazin P by the plant was different with the growth stages of the plant, and the rate of uptake was rapid in early growth stage. Kitazin P penetrated into plant tissues was gradually hydrolyzed to produce O,O-diisopropyl hydrogen phosphorothioate which was converted to diisopropyl hydrogen phosphate, isopropyl dihydrogen phosphate and phosphoric acid. As toluene soluble metabolites, eight spots were detected by thin-layer chromatography, but their percentages in toluene soluble fraction were extremely low as compared with that of Kitazin P. Only two metabolites, dibenzyl disulfide and O,O-diisopropyl O-benzyl phosphorothionate were identified by a gas-liquid chromatography with a flame thermionic detector or a flame photometric detector. Diisopropyl hydrogen phosphorothioate was detected as a persistent metabolite even in rice grains.     

OsGLU1, A Putative Membrane-bound Endo-1,4-ß-D-glucanase from Rice, Affects Plant Internode Elongation

A dwarf mutant glu was identified from screening of T-DNA tagged rice population. Genetic analysis of the T1 generation of glu revealed that a segregation ratio of wild-type:dwarf phenotype was 3:1, suggesting that the mutated phenotype was controlled by a single recessive nuclear locus. The mutated gene OsGLU1, identified by Tail-PCR, encodes a putative membrane-bound endo-1,4-β-D-glucanase, which is highly conserved between mono- and dicotyledonous plants. Mutation of OsGLU1 resulted in a reduction in cell elongation, and a decrease in cellulose content but an increase in pectin content, suggesting that OsGLU1 affects the internode elongation and cell wall components of rice plants. Transgenic glu mutants harboring the OsGLU1 gene complemented the mutation and displayed the wild-type phenotype. In addition, OsGLU1 RNAi plants showed similar phenotype as the glu mutant has. These results indicate that OsGLU1 plays important roles in plant cell growth. Gibberellins and brassinosteroids induced OsGLU1 expression. In rice genome, endo-1,4-β-D-glucanases form a multiple gene family with 15 members, and each may have a distinct expression pattern in different organs. These results indicate that endo-1, 4-β-D-glucanases may play diverse roles in growth and developmental process of rice plants. Hua-Lin Zhou, Si-Jie He: These authors contributed equally to this work     

Studies on γ Globulin of Rice Embryo

All globulin components hitherto found in many species of seeds, α, β, γ and δ globulins, were identified in rice grain by ultracentrifugal experiments and gel-filtration chromatography. Among them, γ globulin was found to occur in high concentration in embryo and bran which were the most active parts in biological functions of rice grain. Then γ globulin was isolated from embryo by gel-filtration chromatography on a Sephadex G-200 column. Purified γ globulin was homogeneous in ultracentrifugal analysis and it was found to be insoluble in cold saline solution. On the other hand, α and β globulins were found to be more concentrated in endosperm with considerable heterogeneity.     

Studies on γ Globulin of Rice Embryo

An improved method has been described for the isolation and purification of γ globulin from rice embryo. The method involves the extraction with phosphate buffer, pH 7.0 and ionic strength 0.1, the fractionation in saline solution of ionic strength 0.31, the removal of nucleic acids by precipitation with ammonium sulfate and the gel filtration chromatography on a Sephadex G-200 column. Although the preparations exhibited homogeneous patterns in sedimentation analysis, the electrophoretic patterns on polyacrylamide gels at pH 8.35 and ionic strength 0.11 exhibited at least two components. Three major components, γ₁, γ₂ and γ₃ globulins, were isolated by ion exchange chromatography on a DEAE Sephadex A-50 column. These components were revealed to be homogeneous in electrophoresis as well as sedimentation. N-Terminal amino acid compositions have also been described.

The molecular weight of γ₁ globulin was determined as 2.0 × 10⁵ by the Archibald method, and the intrinsic viscosity, [η], and the sedimentation coefficient, s_{20, w}^°, were found to be 0.0424 dl/g and 7.26S respectively. These values indicated the large asymmetry of the protein. The protein was composed of 18 residues of hexose, 3 residues of pentose, 6 residues of hexosamine and 1751 residues of amino acids: Lys₅₈, His₄₇, Arg₁₄₈, Asp₁₂₆, Glu₂₇₃, Gly₁₆₁, Ala₁₄₄, Val₁₂₁, Leu₁₀₆, Ile₇₂, Pro₈₃, Ser₁₃₆, Thr₄₈, Hyp₆₈, Cys₁₇, Met₁₆, Tyr₄₄, Trp₈, Phe₇₅ and amide ammonia₁₆₃. The N-terminal amino acid analysis suggested that the protein was composed of ten subunits. The properties and the composition were discussed in comparison with those of the 7S globulin of soybean cotyledon.     

Inhibitory Effects of Cycasin on Germination,Growth and α-Amylase Biosynthesis in Rice Plants

Cycasin, the toxic glycoside of cycad plants, interfered with seed germination and seedling growth of Gramineae, Crucifereae and Leguminosae. The shoots and roots of seedlings showed wilting, chlorosis and necrosis. Rice plants were most sensitive and soybean plants rather tolerant.

Respiration and α-amylase activity were markedly low in the rice seedlings treated with cycasin. Both cycasin and its aglycone, methylazoxymethanol, did not inhibit the activity of α-amylase, but did suppress the formation of α-amylase in rice endosperms. Exogenous gibberellin considerably reversed the inhibition of germination and growth, and the suppression of α-amylase formation caused by these toxins.     

Studies on γ Globulin of Rice Embryo

The secondary structure of γ₁ globulin from rice embryo was investigated by means of optical rotatory dispersion, circular dichroism and infrared spectroscopy. The optical rotatory dispersion curve of the native γ₁ globulin gave a trough at 233mμ with a [m′]₂₃₃ value of ?2,100°, and the Moffitt-Yang plot gave the parameters of a₀= ?237 and b₀= ?20. These data suggest the presence of 3% helix and 38%β structure in the molecule. Circular dichroism exhibits a negative extremum at 218 mμ, giving a [θ]_R value of ?3,730, which suggests the presence of 16°β structure. Infrared spectrum of a thin film of γ₁ globulin showed absorption bands at 695 and 660 cm^?1 with a small hump at 615 cm^?1 characteristic of the β structure, random coil and α helix, respectively. The protein in heavy water exhibits the absorption maximum at 1,630cm^?1 which is also characteristic of the β structure.     

Studies on γ Globulin of Rice Embryo

The molecular weight of γ₃ globulin was determined to be 120,000 daltons by means of both sedimentation equilibrium and gel filtration methods. The protein was composed of 3 identical major and 1 minor subunits, and the molecular weights of them were found to be 35,000 and 13,000 daltons, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major subunit has an arginyl residue as the amino terminal amino acid. The amino acid and carbohydrate composition of γ₃ globulin was determined as follows: Lys₃₇His₃₇Arg₉₂Asp₆₀Glu₁₃₉Gly₁₂₀Ala₈₃Val₇₆Leu₇₀Ile₃₁Pro₅₄Ser₇₇Thr₃₃Cys₁₁Met₉Phe₅₁Tyr₂₆Trp₈ (Amide NH₃)₆₉Hexose₁₅Pentose₄Hexosamine₄. The structure of γ₃ globulin was discussed with comparing that of γ₁ globulin.     

Absorption and Translocation of O-Ethyl S,S-Diphenyl Phosphorodithiolate (Hinosan®) in Rice Plants

Absorption and translocation of (O-ethyl S,S-diphenyl phosphorodithiolate (Hinosan) in rice plants were studied by means of techniques of gas-liquid chromatography, radioautograph and ³²P-radioactivity determination. ³²P-labeled Hinosan was used in the present study. Hinosan on the surface of a leaf and on a glass plate was dissipated in a much slower rate than the other phosphorus pesticides tested. Hinosan was chemically transformed to a small extent after taken up into the plant tissues and largely remained in the local region. Up- and downward translocation of Hinosan occurred slightly in the ride seedlings. Translocation and accumulation of Hinosan were also studied concerning with growth stage of rice plants, such as the seedling, the milk-ripening, and the ripening stage. Accumulation in grains was in most cases found to be less than the limit of determination. Translocation of Hinosan was discussed in relation to physiological conditions of rice plants.     

Identification of QTLs with Additive,Epistatic, and QTL × Seed Maturity Interaction Effects for Seed Vigor in Rice

Seed maturity is a critical process of seed vigor establishment. In this study, one rice population of recombinant inbred lines (RILs) was used to determine the genetic characteristics of seed vigor, including the germination potential (GP), germination rate (GR), germination index (GI), and time for 50 % of germination (T₅₀), at 4, 5, and 6 weeks after heading in 2 years. Significant differences of seed vigor were observed among two parents and RIL population; the heritability of four traits was more than 90 % at three maturity stages. A total of 19 additive and 2 epistatic quantitative trait loci (QTL) for seed vigor were identified using QTL Cartographer and QTLNetwork program, respectively, in 2012, while 16 simple sequence repeat (SSR) markers associated with seed vigor were detected using bulked segregant analysis (BSA) in 2013. The phenotypic variation explained by each additive, epistatic QTL, and QTL × seed maturity interaction ranged from 9.19 to 22.94 %, 7.23 to 7.75 %, and 0.05 to 0.63 %, respectively. Ten additive QTLs were stably expressed in 2 years which might play important roles in establishment of seed vigor in different environments. By comparing chromosomal positions of ten stably expressed additive QTLs with those previously identified, they might be true QTLs for seed vigor; the regions of QTLs for seed vigor are likely to coincide with QTLs for seed dormancy, seed reserve mobilization, low-temperature germinability, and seedling growth. Using four selected RILs, three cross-combinations were predicted to improve seed vigor; 9 to 10 elite alleles could be pyramided by each combination. The selected RILs and the identified QTLs might be applicable for the improvement of seed vigor by marker-assisted selection (MAS) in rice.     

Farmers’ Preference for Rice Traits: Insights from Farm Surveys in Central Luzon,Philippines, 1966-2012

Many modern rice varieties (MVs) have been released but only a few have been widely adopted by farmers. To understand farmers’ preferences, we characterized MVs released in the Philippines from 1966 to 2013 and identified important characteristics of the varieties that were widely adopted in Central Luzon using farm surveys conducted in 1966–2012. We found that farmers adopt MVs that are high yielding, mature faster, and have long and slender grains, high milling recovery, and intermediate amylose content. The amylose content of adopted varieties has been declining, suggesting value in developing softer rice. To have a high potential for adoption, new MVs should have characteristics within the ranges of values observed for the adopted MVs. In addition, new MVs should have higher head rice recovery, less chalky grains, and better resistance to pests and diseases. Most MVs released in 2005–2013 compared poorly in these three traits. To reduce the risk of severe outbreaks, broad spectrum resistance should be incorporated into new MVs. This analysis of five decades of farm surveys provides insights into the varietal characteristics preferred by farmers which could contribute to the establishment of a product profile for developing improved MVs that are more targeted and, hence, would have high potential for adoption by farmers in Central Luzon and similar areas. We recommend a similar analysis be done in other major rice growing regions to aid the development of MVs that are more responsive to farmers’ needs and preferences.     

Nitrogen and α-Ketoglutaric Acid Application Modulate Grain Yield,Aroma, Nutrient Uptake and Physiological Attributes in Fragrant Rice

Journal of Plant Growth Regulation - The present study was conducted to assess the effects of nitrogen (N) and α-ketoglutaric acid application morpho-physiological attributes, aroma...     

Four Rice Seed cDNA Clones Belonging to the α-Amylase/Trypsin Inhibitor Gene Family Encode Potential Rice Allergens

Four rice seed proteins encoded by cDNAs belonging to the α-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial celllysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.     

Tracking Se Assimilation and Speciation through the Rice Plant – Nutrient Competition,Toxicity and Distribution

Up to 1 billion people are affected by low intakes of the essential nutrient selenium (Se) due to low concentrations in crops. Biofortification of this micronutrient in plants is an attractive way of increasing dietary Se levels. We investigated a promising method of Se biofortification of rice seedlings, as rice is the primary staple for 3 billion people, but naturally contains low Se concentrations. We studied hydroponic Se uptake for 0–2500 ppb Se, potential phyto-toxicological effects of Se and the speciation of Se along the shoots and roots as a function of added Se species, concentrations and other nutrients supplied. We found that rice germinating directly in a Se environment increased plant-Se by factor 2–16, but that nutrient supplementation is required to prevent phyto-toxicity. XANES data showed that selenite uptake mainly resulted in the accumulation of organic Se in roots, but that selenate uptake resulted in accumulation of selenate in the higher part of the shoot, which is an essential requirement for Se to be transported to the grain. The amount of organic Se in the plant was positively correlated with applied Se concentration. Our results indicate that biofortification of seedlings with selenate is a successful method to increase Se levels in rice.